1. Field of the Invention
The present invention relates to G protein-coupled receptor (GPCR) microarrays on porous substrates for structural or functional analyses of GPCRs, and methods of preparing porous substrate surfaces for receiving membranes that comprise GPCRs.
2. Background of Related Art
GPCRs are the single most important class of drug targets—approximately 50% of current drug targets are membrane bound. Despite the large number of GPCR targets and a wide variety of technologies for screening against GPCRs, no methods were available for screening against multiple GPCRs simultaneously. The GPCR microarray technology has been investigated (Fang et al., “G Protein-coupled Receptor Microarrays for Drug Delivery” Drug Discovery Today, Vol. 8, No. 16, August 2003, pp. 755-761; Bieri et al., “Micropatterned Immobiliztion of a G Protein-coupled Receptor and Direct Detection of G Protein Activation,” Nature Biotechnology, Vol 17, November 1999, pp. 1105-1108; Pierce et al., “Seven-Transmembrane Receptors” Molecular Cellular Biology, Vol. 3, September 2002, pp 639-650) and their use has been demonstrated for the multiplexed screening of compounds, see for example U.S. Patent Application Publication Nos. 2002/0019015 and 2002/0094544, the entire disclosures of which are hereby incorporated by reference.
The arrays were obtained on flat “2D” glass slides coated with γ-aminopropylsilane (GAPS) and other materials including epoxypropylsilane. Most assay development has focused on “binding assays” that provide information about how much of a compound is bound to a receptor at a particular concentration; based on this information, the affinity of the compound for the receptor can be obtained.
A large fraction of GPCR screening assays—so-called “functional assays”—are based on determining whether the GPCR gets activated as a result of compound binding. The information can be used to classify compounds as agonists, partial agonists, antagonists or inverse agonists. Moreover, functional assays are essential for investigating “orphan” GPCRs, some of which may turn out to be key drug targets. Orphan GPCRs are those without known ligands, which preclude the use of competition assays employing known labeled ligands. Functional assays can be both cell-based and biochemical in nature; cell-based assays are currently the method of choice for functional assays. Cell based assays include reporter gene assays, β-arrestin and GPCR-GFP translocation assays (i.e., receptor internalization and endosome formation). Methods for monitoring the activation of GPCRs by non-cell based assays are mostly limited to monitoring GTP-GDP exchange at the GPCR associated Gα protein using labeled GTP analogues (e.g., 35S-GTP γS or Eu-GTP). These functional assays are “homogenous” assays, that is, the receptor and the GTP analogue mixed with or without a compound of interest are in solution over the duration of the assay; these assays are then subject to filtration using a filter microplate so that the labeled GTP can be removed by filtration, and only the bound GTP analog molecules can be quantified and the effect of compound on the binding of GTP analog can be examined which can be used to classify the action of compound on the receptors (i.e., non-binder, or antagonist, or agonist, etc).
Limited success has been encountered with the use of fluorescent-dye labeled GTP-γS for functional assays on 2D GAPS surfaces, although functional assays employing radioactively labeled 35S-GTPγS have been successfully carried out on these surfaces. However, the relatively poor reproducibility of these functional assays limits their applications of GPCR microarrays for compound screening. Moreover, the use of non-radioactive labels is preferred because of safety issues. Europium-labeled GTP (Eu-GTP) (Perkin Elmer Life Science, Boston, Mass.) has been developed as an alternative to 35S-labeled-GTP, and has been successfully demonstrated their use in functional assays carried out in solution in combination with filter-plates. Realization of Eu-GTP binding assays for GPCR microarrays on porous substrates would greatly benefit their applications for compound screening.
With regard to the production and use of GPCR microarrays, the G protein coupled receptor (GPCR) microarrays are unique in that they require immobilization of both the protein targets and the lipid membrane in which they are embedded (Fang et al., “Membrane Protein Microarrays,” J. American Chemical Society, Vol. 124, 2002, pp. 2394-2395; Fang et al., “G-Protein Coupled Receptor Microarrays,” Chembiochem., Vol. 3, 2002, pp. 987-991). Moreover, the confined proteins should be in their correctly folded conformations. Different types of surfaces have been proposed that meet these requirements (Hennestal et al., “Pore Spanning Lipid Bilayers Visuallized by Scanning Force Microscopy,” J. American Chemical Society, Vol. 122, 2000, pp. 8085-8086; Cremer et al., “Formation and Spreading of Lipid Bilayers on Planar Glass Supports,” J. Physical Chemistry B, Vol. 103, 1999, pp. 2554-2559; Theato et al. “Formation of Lipid Bilayer on a New Amphiphiic Polymer Support,” Langmuir, Vol. 16, 2000, pp. 1801-1805; Majewski et al., “Structural Studies of Polymer-Cushiond Lipid Bilayers,” J. Biophysical Journal, Vol. 75, 1998, pp. 2363-2367.
Conventional methods for fabricating solid supported membranes exploit gold-thiol, capping of OH-groups by silanes, and electrostatic interactions. The resulting membranes exhibit limited long-term stability due to the lipid loss into the solution when remained in aqueous solutions (Fang and Yang, “The Growth of Bilayer Defects and the Induction of Interdigitated Domains in the Lipid-Loss Process of Supported Phospholipid Bilayer,” Biochim. Biophys. Acta, Vol. 1324, 1997, pp. 309-319), but their close proximity to the solid surface (typically 0.2-2 nm) limits lateral lipid mobility. Since a membrane-surface separation of at least 1 to 5 nm (preferably at least 2 to 10 nm) is usually required to preserve the biological functions of the membrane proteins associated with the membranes, several approaches have been employed to extend the membrane surface distance, such as the use of lipids with long hydrophilic spacers, the inclusion of polymer cushions between substrate and membrane, and the use of patterns with varied thiol-components that increase lateral mobility and free volume.
A functional GPCR assay is possible if both GPCR terminals are accessible and bioactive. Suspended membranes have been developed on the basis of membranes spanning the pores of porous alumina substrates (Hennestal et al., supra). When the membrane spans the pores there are no issues with steric congestion on either side of the receptor.
A method has been proposed which makes use of “contact printing” to deposit a binding chemistry, such as a moderately positively charged coating, only onto the top surface of a porous substrate. The contact printing includes impregnating a flat polymer stamp with a solution containing the active molecules and brings it in conformal contact with the porous substrate. This effectively transfers the active molecules only onto the top surface of the substrate.
Further, it is also known to perform functional assays for G-Protein Coupled Receptors (GPCRs) in commercially available 96 well plates using a time-resolved fluorometric assay based on GDP-GTP-Eu-labeled exchange on GPCR. The activation of receptors by agonists is made in solution inside wells. The activation signal is detected on the porous bottom of wells where the activated receptor of the GPCR is retained after filtration (see, for example, the DELFIA GTP-binding kit from Perkin Elmer Inc.).
G-protein coupled receptor (GPCR) microarrays are unique in that they allow immobilizing both the protein targets and the lipid membrane in which they are embedded before activation. One advantage of this technique is to use small amounts of expensive receptors and to study several receptors simultaneously in the same well. However, the confined protein should be in their correctly folded conformations. Different types of surfaces have been proposed that meet these requirements as discussed above.
Therefore, it can be realized that effective GPCR microarrays for use in functional assays, e.g., employing particular GTP analogues, are needed. Additionally, a simple method of selecting the appropriate porous substrate for receiving a membrane, enhancing the immobilization of the membrane on the porous support, as well as a simple method of fixing a membrane on the porous support, are needed.